Identification and characterization of extrachromosomal circular DNA in maternal plasma

PNAS January 21, 2020

Significance

We observed the presence of extrachromosomal circular DNA  (eccDNA) in the plasma of pregnant women. We found that the plasma  eccDNA molecules were longer than their linear counterparts. Among such  eccDNA molecules, those of fetal origin were shorter than those of  maternal origin. Characteristic dual-repeat patterns of eccDNA junctions might shed light on their possible generation mechanisms and provide  them with distinctive signatures over linear cell-free DNA. Furthermore, the closed circular structure of eccDNA might allow resistance to  exonucleases and thus higher stability of these molecules over their  linear counterparts. These features of eccDNA provide opportunities for  research and biomarker development. This work represents an example in  the nascent field of plasma DNA topologics.

Abstract

We explored the presence of extrachromosomal circular DNA  (eccDNA) in the plasma of pregnant women. Through sequencing following  either restriction enzyme or Tn5 transposase treatment, we identified  eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with  distinct 10-bp periodicity observed throughout the size ranges within  both peaks, suggestive of their nucleosomal origin 这些eccDNA分子显示出双峰大小分布，峰值分布在〜202和〜338 bp，在两个峰的整个大小范围内观察到明显的10 bp周期性，表明它们的核小体起源. Also, the  predominance of the 338-bp peak of eccDNA indicated that eccDNA had a  larger size distribution than linear DNA in human plasmaeccDNA的338bp峰占优势，表明eccDNA在人血浆中的分布比线性DNA大。. Moreover,  eccDNA of fetal origin were shorter than the maternal eccDNA胎儿来源的eccDNA比母亲的eccDNA短. Genomic  annotation of the overall population of eccDNA molecules revealed a  preference of these molecules to be generated from 5′-untranslated  regions (5′-UTRs), exonic regions, and CpG island regions. Two sets of  trinucleotide repeat motifs flanking the junctional sites of eccDNA  supported multiple possible models for eccDNA generation. This work  highlights the topologic analysis of plasma DNA, which is an emerging  direction for circulating nucleic acid research and applications.

The fragmentation patterns of cell-free DNA (cfDNA) in human plasma is an area of intense research interest (1⇓⇓–4). Recent studies on the size distributions (1), end locations (5), and end motifs (3) revealed that these fragmentation patterns in cfDNA bore relationships  with their tissues of origin. In pregnancy, fetal-derived plasma DNA  (mainly of placental origin) were observed to be linear fragments of DNA that were shorter than the maternal-derived (mainly of hematopoietic  origin) DNA (1, 5, 6). In cancer, tumor-derived cfDNA were detected with smaller sizes and  preferred end coordinates from those derived from nonmalignant cells (7⇓–9). Diagnostic applications had been demonstrated for using the  fragmentation patterns of plasma DNA in noninvasive prenatal testing and cancer testing (5, 6, 10⇓–12). However, the above-mentioned studies predominantly focused on linear  DNA fragments in plasma. We have recently demonstrated that there were  different topologic forms (i.e., linear as well as circular) of plasma  mitochondrial DNA (mtDNA) (13). Of interest, we observed that circular mtDNA molecules were  predominantly of hematopoietic origin, whereas the liver-derived ones  were predominantly linear.我们观察到圆形mtDNA分子主要来自造血，而肝脏衍生的分子主要是线性的。

In this work, we explored plasma DNA molecules originated  from the genome that were of other topological forms. In particular, we  focused on extrachromosomal circular DNA (eccDNA) molecules in the  plasma of pregnant women. This special form of DNA molecules had  previously been observed across different species of organisms from  yeast to mouse (14, 15). The sizes of eccDNA varied widely, ranging from dozens of bases to  hundreds of thousands of bases, with the majority of them being smaller  than 1,000 bp (15, 16). These eccDNA molecules were found to be enriched from genomic regions with high gene densities and GC contents (15, 17). The presence of eccDNA in human and murine plasma had also been reported (18, 19). However, there are no published data on eccDNA in the plasma of  pregnant women. Our first goal in this work was therefore to investigate whether a fetus might release eccDNA into the plasma of its pregnant  mother, analogous to the presence of linear fetal DNA in the plasma of  pregnant women (1, 20). Second, we compared the size profiles between maternal and fetal  eccDNA. Last, we explored nucleotide motif signatures flanking the  eccDNA junctional sites in the hope of gaining insights into eccDNA  generation mechanisms.

Results

Identification of eccDNA in Plasma by MspI Digestion.

We analyzed plasma DNA samples from five cases of third-trimester pregnancy by MspI digestion. Circular DNA molecules in plasma were first enriched by  exonuclease V (exo V) digestion of the background linear DNA. MspI restriction enzyme was then used to linearize the remaining circular  DNA, followed by library construction and next-generation sequencing.  The workflow of eccDNA detection from plasma DNA is described in Fig. 1, from which we developed bioinformatics algorithms for eccDNA identification and downstream analyses (see details in Materials and Methods). The “junction” indicated the position where two ends of a genomic sequence were ligated, forming a DNA circle.

Fig. 1. Workflow of eccDNA identification.  eccDNA generated from the genome would possess a start (blue) and an end (red) position, which were ligated to form a junctional site. eccDNA  molecules in the plasma were cleaved by MspI restriction  enzyme, followed by library construction procedures. Paired-end  sequencing of the DNA libraries were performed on the Illumina HiSeq  1500/2500 platforms. Sequencing reads were aligned to the reference  genome, and algorithms were developed to identify eccDNA. Sequencing  reads meeting the four criteria of eccDNA identification (see Materials and Methods for details) were assigned as eccDNA fragment reads.

The plasma eccDNA counts of each sample was normalized as  eccDNA per million mappable reads (EPM). The number of mappable reads of each sample used in this calculation was the total number of reads  mapped to both chromosomal and mtDNA in that sample. We first confirmed  the efficiency of exo V in enriching eccDNA molecules from plasma  samples. We compared the EPM values of case 13007 with and without exo V treatment, followed by MspI digestion. We observed a 10,014-fold increase in EPM value after exo V treatment (EPM [exo V + MspI]: 6,409; EPM [MspI only]: 0.64). Thus, exo V treatment could significantly enrich eccDNA molecules. For the five pregnancy cases examined by the MspI approach (exo V + MspI), the median EPM value was 1,462 (range, 844 to 6,409). We further  plotted the plasma eccDNA size distributions and compared them with  their linear counterparts from the same subjects. Size profiles of these five cases showed that the linear DNA and eccDNA molecules in plasma  had distinct size distributions (Fig. 2A). The linear plasma DNA showed a predominant size peak at ∼166 bp with a  10-bp periodic pattern in molecules smaller than 166 bp. Such a size  distribution is in concordance with previous reports on linear plasma  DNA (1, 21, 22). On the other hand, eccDNA molecules detected from MspI-treated samples showed two major peaks at 202 and 338 bp. The 338-bp peak was  around 10 to 30 times more pronounced than the 202-bp peak as indicated  by their areas under the curves.按照曲线下的面积计算，338 bp的峰值比202 bp的峰值大10到30倍。 The predominant dinucleosomal size  signature of plasma eccDNA observed here was in concordance with  previous reports of plasma samples from nonpregnant subjects此处观察到的血浆eccDNA的主要二核小体大小特征与先前来自非妊娠受试者的血浆样品报道一致 (15, 18, 23). Moreover, there was a distinct 10-bp periodicity throughout the size  ranges within each of the peaks. Notably, such small peaks at 10-bp  intervals were of almost identical sizes among different cases. The  eccDNA size profiles of individual cases and the sizes of each small  peak are plotted in SI Appendix, Fig. S1.

Fig. 2.

eccDNA identification by the restriction enzyme (MspI) approach. Plasma samples of five pregnancy cases were analyzed. (A) Size distributions of linear (blue) and eccDNA (red) in the plasma. (B and C) Plots of size distributions of maternal- and fetal-derived plasma eccDNA, respectively. (D) Cumulative frequency plots of maternal- (blue) and fetal-derived (red) eccDNA in plasma.

Detection and Analysis of Fetal eccDNA in Maternal Plasma.

It had previously been shown that fetal-derived linear  plasma DNA molecules were generally shorter than the maternal-derived  molecules (1, 24). To compare the maternal- and fetal-specific eccDNA in plasma, we  classified eccDNA fragments carrying fetal-specific single-nucleotide  polymorphism (SNP) alleles as fetal-derived eccDNA, and the ones that  carried maternal-specific SNP alleles as maternal-derived eccDNA. Fig. 2 B and C show the size distributions of maternal- and fetal-derived eccDNA of the five pregnancy cases (MspI-treated), respectively. Both maternal and fetal eccDNA exhibited two major peaks  at ∼202 and ∼338 bp, with both peaks being sharper for the fetal  population 母体和胎儿的eccDNA都在〜202和〜338 bp处出现了两个主要峰，这两个峰对于胎儿群体而言都更为尖锐。 Furthermore, both maternal and fetal eccDNA plots showed a  10-bp periodicity in proximity to both peaks. Fig. 2D shows that the cumulative frequency curve of fetal-specific eccDNA was  located on the left of the maternal-specific curve. Hence, fetal-derived eccDNA molecules were generally shorter than the maternal-derived ones. Such size differences between maternal- and fetal-derived eccDNA were  thus consistent with those of their linear counterparts (1, 24). Also, the fetal DNA fractions deduced from eccDNA showed a positive  correlation with those deduced from linear DNA from the same samples (SI Appendix, Fig. S2).